Origin and chromatin remodeling of young X/Y sex chromosomes in catfish with sexual plasticity

ABSTRACT Assembly of a complete Y chromosome is a significant challenge in animals with an XX/XY sex-determination system. Recently, we created YY-supermale yellow catfish by crossing XY males with sex-reversed XY females, providing a valuable model for Y-chromosome assembly and evolution. Here, we assembled highly homomorphic Y and X chromosomes by sequencing genomes of the YY supermale and XX female in yellow catfish, revealing their nucleotide divergences with only less than 1% and with the same gene compositions. The sex-determining region (SDR) was identified to locate within a physical distance of 0.3 Mb by FST scanning. Strikingly, the incipient sex chromosomes were revealed to originate via autosome–autosome fusion and were characterized by a highly rearranged region with an SDR downstream of the fusion site. We found that the Y chromosome was at a very early stage of differentiation, as no clear evidence of evolutionary strata and classical structure features of recombination suppression for a rather late stage of Y-chromosome evolution were observed. Significantly, a number of sex-antagonistic mutations and the accumulation of repetitive elements were discovered in the SDR, which might be the main driver of the initial establishment of recombination suppression between young X and Y chromosomes. Moreover, distinct three-dimensional chromatin organizations of the Y and X chromosomes were identified in the YY supermales and XX females, as the X chromosome exhibited denser chromatin structure than the Y chromosome, while they respectively have significantly spatial interactions with female- and male-related genes compared with other autosomes. The chromatin configuration of the sex chromosomes as well as the nucleus spatial organization of the XX neomale were remodeled after sex reversal and similar to those in YY supermales, and a male-specific loop containing the SDR was found in the open chromatin region. Our results elucidate the origin of young sex chromosomes and the chromatin remodeling configuration in the catfish sexual plasticity.


INTRODUCTION
The XX/XY chromosome system is the most common sex-determination system in vertebrates. In mammals, sex is usually determined by the major sex-determining region (SDR) on the Y chromosome [1]. Complete and high-quality Y-chromosome sequences are essential for studying the evolution and mechanism of sex determination [2]. Among most sequencing projects, individuals of homogametic sex (XX females) are usually chosen for genome sequencing and assembly, whereas whole Y-chromosome sequences are assembled mainly in a handful of XY-male mammals through a time-and labor-intensive clone-by-clone approach [3][4][5][6][7], as well as in YY-supermale fishes generated by artificial sex-reversal technology [8,9], and in XYmale fish by telomere-to-telomere assembly [10]. It is widely accepted that sex chromosomes evolve from a pair of autosomes, one of which acquires a sex-determining locus. Next, the progressive evolutions of recombination suppression though C The Author(s) 2022. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. chromosome rearrangements lead to the formation of heteromorphic X and Y chromosomes [11][12][13][14]. The prevailing models of sex-chromosome origin and evolution are mainly based on the stable and highly differentiated Y chromosomes in mammals. However, sex-chromosome evolution and the recombination suppression mechanism are more complex than previously expected and a broader understanding of vertebrate sex chromosomes is hindered by the limited number and no availability of high-quality Y-chromosome sequences [15].
In contrast to the heteromorphic sex chromosomes of mammals, homomorphic sex chromosomes with poor differentiation are observed in many lower vertebrates, including most fish species, which provide a good model to study the initial stages of sex-chromosome evolution [16,17]. During the process of sex-chromosome evolution, multiple sex-chromosome constitutions have been frequently observed in fishes and reptiles, resulting from the fusion of sex chromosomes and autosomes, as whole-chromosome fusions are more frequent on sex chromosomes than in autosomes [18][19][20]. Recently, it was proposed that autosome-autosome fusions occurred before the recruitment of the fused autosomes as sex chromosomes [21]. However, there is no available model to investigate the evolutionary mechanism of sex-chromosome formation via autosome-autosome fusion.
Sex determination in fish and amphibians is a plastic process that can be modulated by both genetic and environmental factors [22,23]. The most common form of environmental sex determination (ESD) in fish and amphibians is temperature-dependent sex determination (TSD), which is controlled by epigenetic modifications, such as genomic DNA methylation [24] and histone modification [25,26]. It is widely recognized that epigenetic modifications can modulate chromatin architecture and dynamics, which play critical roles in gene-expression regulation, cell differentiation and developmental processes [27,28]. Temperature-sensitive and temperature-insensitive populations exist simultaneously in some fish and amphibian species, and transitions between genetic sex determination (GSD) and TSD have been reported in gibel carp, yellow catfish, Nile tilapia and bearded dragon [29][30][31][32][33][34] and Y-autosome fusion was revealed to generate neo-Y chromosomes in fish and amphibians [18,35]. However, it remains unclear whether the chromatin architecture of poorly differentiated Y chromosomes contributes to sex determination and male-biased gene expression.
Recently, YY-genotype male yellow catfish, channel catfish and southern catfish have been generated via the integration of hormonally induced sex-reversal technology and sex-linked marker identification [8,9,36,37], providing a unique research model for studying the structure and evolution of sex chromosomes in catfishes. Sexually reversed XX-neomale yellow catfish were artificially produced by using an aromatase inhibitor, letrozole, and artificially induced sex reversal leads to a transition from GSD to TSD in yellow catfish [30]. In this study, we assembled and compared X-and Y-chromosome sequences of yellow catfish by sequencing the genomes of XX females and YY supermales to explore the origin and evolution of homomorphic X and Y chromosomes with an XX/XY sex-determining system. Strikingly, sex chromosomes in yellow catfish were revealed to originate from autosome-autosome fusion. We further compared the chromatin architectures of the X and Y chromosomes in XX-female, XX-neomale and YYsupermale yellow catfish and found the potential roles of the 3D chromatin architecture in fish sex determination and sexual plasticity.

Identification of sex chromosomes by bacterial artificial chromosomefluorescence in situ hybridization (BAC-FISH)
Sex-chromosome-linked markers and BAC clones containing sex-chromosome-linked markers have been identified in previous work [36]. To identify the sex chromosomes of yellow catfish, we performed FISH on three different genotypes of yellow catfish (XX female, XY male and YY supermale) with BAC DNA as a probe. As shown in Supplementary Fig. 1A, two signal loci were observed on a pair of homologous chromosomes in metaphase cells of XY males. Based on the hybridization signals of sexchromosome-linked BAC localization, we identified the sex chromosomes of yellow catfish. A similar localization and the same number of hybridization signals were observed in the metaphase cells of XX females and YY supermales (XX: Supplementary Fig.  1B; YY: Supplementary Fig. 1C).
The karyotypic analysis of somatic cells indicates that the chromosome number of yellow catfish is 2n = 52. According to the identified sexspecific markers and chromosome-level assembly of the yellow-catfish genome [36,38], Chromosome 2 is the sex chromosome and has the second largest size among all chromosome pairs ( Supplementary  Fig. 1D). We infer that the sex chromosomes are a pair of submetacentric chromosomes based on their morphological features along with the hybridization signals of sex-linked markers on their two long arms ( Supplementary Fig. 1E). However, in XY-genotype metaphases, the BAC DNA probe could not directly distinguish the X and Y chromosomes via the FISH method. The highly homomorphic structure of this sex-chromosome pair may be the reason why we could not distinguish them using traditional cytogenetic methods. This also suggests that this sex-chromosome pair is still in the initial stage of evolution.

Accurate genome assembly of YY supermale and XX female
Whole-genome sequencing and assembly were performed on an YY supermale and XX female to explore their genomic differences in yellow catfish. A YY supermale was chosen for genome sequencing using a combination of several technologies, including single-molecule real-time sequencing (PacBio RS II), paired-end sequencing (Illumina HiSeq 2000) and chromatin conformation capture (Hi-C). Finally, these sequences were assembled into 1590 contigs, with a contig N50 of 2.95 Mb and a scaffold N50 of 27.04 Mb. The total assembled genome sequence of YY supermale was 710 Mb, while the total length of the anchored contigs was ∼706 Mb. A total of 94.7% of the yellow catfish genes completely matched the Benchmarking Universal Single-Copy Orthologs (BUSCO) set (Supplementary Table 1). In addition, the XX genome [38] was reassembled and improved according to the same assembly methods as for the YY genome. The XX genome sequences were finally assembled into 1335 contigs, with a contig N50 of 3.16 Mb and a scaffold N50 of 27.16 Mb. The total assembled genome sequence of the XX yellow catfish was 710 Mb (Supplementary Table 1). A total of 95.0% of the XX yellow-catfish genes completely matched the BUSCO set and the assembly metrics showed considerable improvement relative to the previous assembly [38]. To further evaluate assembly completeness, the genomic sequences of XX and YY yellow catfish were directly aligned with LAST and extremely high sequence similarity was found ( Supplementary Fig. 2).

Subtle divergence of repetitive sequence subfamilies between X and Y chromosomes
We next scrutinized the sequence differences between the X and Y chromosomes to reveal the potential sex-determination genes. The assembled X chromosome was 43.5 Mb in length and composed of 39 contigs, while the assembled Y chromosome was 43.2 Mb and composed 48 contigs (Supplemen-tary Table 2). The alignment of the X and Y chromosomes showed >99% sequence identity. In addition, we found highly similar distributions of the GC contents and repeat contents of the X and Y chromosomes ( Supplementary Figs 3 and 4). A total of 1427 genes were annotated on both the assembled Y chromosome and X chromosome, and the gene content was completely shared between the X and Y chromosomes (Fig. 1A). In an attempt to discover whether different evolutionary strata existed on the yellow-catfish sex chromosomes, as observed in some model fish species, including three-spined stickleback [39] and spotted knifejaw [40], we estimated non-synonymous site divergence (Ka), synonymous site divergence (Ks) and Ka/Ks between the X and Y chromosomes but did not find any regions with significant differences in these parameters .
We further analysed the repetitive sequence divergence between the X and Y chromosomes. A total of 39.88% of the X chromosome and 39.48% of the Y chromosome consisted of repeat sequences, which was comparable to the rates among autosomes (39.37%-51.38%). The repetitive elements identified on the X and Y chromosomes were mostly DNA transposons (17.88% and 18.02%, respectively), long terminal repeat (LTR) retrotransposons (LTRs, 7.62% and 6.59%, respectively) and long interspersed elements (LINEs, 5.64% and 5.97%, respectively) (Fig. 1B). However, the classification of repetitive DNA sequences into subfamilies revealed that several subfamilies of DNA transposons (Crypton, Zator, Ginger-1, hAT and hAT-hAT5), LINE/R2 elements and short interspersed nuclear elements (SINEs) have specifically accumulated on the Y chromosome relative to the rest of the genome. In contrast, some subfamilies of DNA transposons (IS3EU, Crypton-A, Dada, hAT-Ac and Maverick) and LTRs (ERV1 and Gypsy) showed a relatively lower content on the Y chromosome (Fig. 1C). Additionally, we found obvious differences in the distributions of DNA/hAT, DNA/Zator, DNA/Crypton-A, SINE and LINE/CR1 elements between the X and Y chromosomes ( Supplementary  Fig. 8). The analysis of the sequence divergence of individual families from the inferred consensus sequences revealed recent changes on the X and Y chromosomes. Several DNA transposon subfamilies (Crypton/A, Kolobok and Merlin) showed higher divergence on the X chromosome, while higher divergence of other DNA transposons (Maverick, IS3EU and hAT/Blackjack) and LTRs (ERVK and Gypsy) was observed on the Y chromosome ( Supplementary Fig. 9). Taken together, these results indicate that subtle divergence of repetitive sequence subfamilies have occurred on the X and Y   chromosomes, suggesting that they are still in a very early stage of sex-chromosome differentiation.

Identification of the SDR
Pooled sequencing reads from the genomic DNA of 20 XX females and 20 YY supermales were mapped to the XX genome to further characterize genomic regions enriched for sex-biased signals (Supplementary Table 3). We obtained a large number of variants (3 476 961 single-nucleotide polymorphisms [SNPs] and 1 359 630 indels) and searched for the variants that were not only fixed in the XX-female pool but were also homozygous mutants in the YY-supermale pool. As a result, 3001 malespecific SNPs and 2285 male-specific indels were identified across the genome, 1809 and 714 of which were concentrated within an ∼400-kb region on Chromosome 2, and this region showed an increase in the fixation index (F ST ) relative to other regions of the sex chromosomes or autosomes ( Fig. 2A). To further confirm these sex-specific variants, we collected additional 19 XX females and 19 YY supermales from another two families and performed re-sequencing (Supplementary Table 3 between these two batches of samples, a number of sex-antagonistic mutations including 714 malespecific SNPs and 248 male-specific indels were identified, all of which were located on Chromosome 2. In addition, the vast majority of these SNPs (617 of 714) and indels (199 of 248) were within a physical distance of 0.3 Mb ( Fig. 2B and Supplementary Fig. 10). The BAC DNA sequence containing sex-chromosome-linked markers was consistently located in this region, suggesting that it should be the SDR of yellow catfish (Fig. 2B). We identified four synteny blocks between the X and Y chromosomes, which covered the entire sex chromosomes. The SDRs of the X and Y chromosomes were located in the largest synteny block and shared the same gene contents in the same order, without any chromosome inversions or translocations (Fig. 2C).
The 487 male-specific SNPs in this region were specifically located in 11 genes, namely nfia, tm2d1, pfpdz1, pdzk1ip1, tal1, ier5, stx6, kiaa1614, plekhb2, vps4b and lama3, with 375 (77.6%) of these malespecific SNPs being located in pfpdz1. Furthermore, only five genes had Y-specific SNPs on the exons, including lama3, vps4b, kiaa1614, tal1 and pfpdz1 (Supplementary Table 4). During the period of sex determination and differentiation in yellow catfish, the mRNA expressions of nfia, pfpdz1, tal1, stx6 and kiaa1614 in the testis of XY males were higher than in the ovary of XX females ( Supplementary Fig. 11), whereas the expression of pdzk1ip1 was very low and could not be detected. By analysing the distribution of transposable elements on pfpdz1X and pfpdz1Y, three Y-specific DNA transposons were identified, including two DNA/CACTA and one DNA/hAT transposons ( Supplementary Fig. 12A). For the retrotransposons, there are a number of Yspecific LTRs and one Y-specific LINE (Supplementary Fig. 12B).

Origin and evolution of the young sex chromosomes by autosome-autosome fusion
Recently, chromosome-level genome assemblies and sex-chromosome-linked markers of channel catfish (Ictalurus punctatus), southern catfish (Silurus meridionalis) and redtail catfish (Mystus wyckioides) have been reported [9,[41][42][43][44]. Along with the chromosomal assemblies of the XX and YY yellow-catfish genomes, we were motivated to explore origin and evolution of the sex chromosomes. All four of these species belong to Siluriformes, among which the southern catfish belongs to the family Ictaluridae, channel catfish belongs to the family Siluridae and redtail catfish and yellow catfish both belong to the family Bagridae. Based on protein sequence similarity, chromosomal collinear synteny among southern catfish, redtail catfish, channel catfish and yellow catfish was evaluated (Fig. 3A). All three fish species other than yellow catfish have 29 pairs of chromosomes (2n = 58) and the chromosomes of these three species are highly syntenic to each other, indicating relatively conserved karyotypes in Siluriformes. The sex chromosome of channel catfish (Chr4) shares homology with Chr13 of yellow catfish, while the sex chromosomes of southern catfish (Chr24) and redtail catfish (Chr26) are homologous to Chr18 of yellow catfish. Notably, the sex chromosomes of yellow catfish, redtail catfish and channel catfish share no homology, while southern catfish shows the same sex chromosome as redtail catfish (Fig. 3A). Due to the closer relationship between yellow catfish and redtail catfish than the other species, we performed interchromosomal rearrangement analysis between them. We observed several chromosome fissions or fusions between yellow catfish and redtail catfish, including large interchromosomal rearrangements on four chromosomes, namely Chr1, Chr2, Chr7 and Chr9, of yellow catfish ( Supplementary Fig. 13). Surprisingly, we found that the sex chromosomes of yellow catfish were probably derived from the fusion of two autosomes that did not undergo interchromosomal rearrangements in the other three species (Supplementary Fig. 14). Synteny analysis between yellow catfish and electric eel (belonging to Gymnotiformes, which is thought to be the sister group to Siluriformes [45,46]) further confirmed the existence of a fusion site at ∼19 Mb in the X/Y chromosomes of yellow catfish ( Supplementary Fig. 15).
In addition, we found that there were many more intrachromosomal rearrangements (inversions and translocations) in the yellow-catfish sex chromosomes than in any of the autosomes by sequence alignment with redtail catfish (Fig. 3B). Therefore, we examined the sequence synteny between the X chromosome of yellow catfish and the two corresponding autosomes of redtail catfish. The vast majority of the observed intrachromosomal rearrangements occurred in a highly rearranged region (HRR) in the downstream half of the fusion site and the SDR was located near the middle of the HRR (Fig. 3C). In addition, we observed that the recombination rate of the yellow catfish sex chromosomes was low near the fusion site and close to 0 in the SDR, consistently with the theory of sex-chromosome evolution [47,48]. Additionally, we annotated the putative centromeric region by searching the most abundant satellite sequences and found that this region consisted of an inversion and showed a low recombination rate (Fig. 3C). The sequence alignments between the sex chromosomes of yellow catfish and the corresponding autosomes of channel catfish and southern catfish revealed the same results ( Supplementary Fig. 16). Due to the high ratio of translocations and inversions, we speculated that the HRR may have the potential to form a new stratum. By examining transcriptomic expression profiles in gonads of XX-female and YY-supermale yellow catfish, 363 male-biased and 235 female-biased genes were identified on the sex chromosomes, of which 93 male-biased and 78 female-biased genes were located in the HRR. The male-biased genes in the HRR showed significantly higher upregulation compared to other regions of sex chromosomes (Pvalue = 0.02365, Wilcoxon test), while the femalebiased genes did not (P-value = 0.3068, Wilcoxon test) (Fig. 3D).

Diverse 3D chromatin architecture on X and Y chromosomes
Although the X and Y chromosomes of yellow catfish showed high sequence identity, their genomic DNA could be packaged into different 3D chromatin architectures because of different repeat contents or transcription factor binding sites shaping the chromatin architecture. To test this hypothesis, we inferred genome-wide chromatin interaction frequencies by performing Hi-C experiments in gonad cells of XX females and YY supermales and obtained ∼463 million and ∼459 million read pairs, respectively. After filtering, a total of 225 million and 205 million valid read pairs were produced in the XX-female and YY-supermale gonad cells, respectively. The generated contact maps showed obvious differences, with a Pearson correlation of 0.88 ( Fig. 4A and Supplementary Fig.  17). Seventy-one and 42 topologically associated domains (TADs) were identified on the X chromosome and Y chromosome, respectively. We found that only one TAD shared the same boundaries on the X and Y chromosomes. The TADs on the Y chromosome were larger than those on the X chromosome (P-value = 4.884 × 10 −7 , Wilcoxon Natl Sci Rev, 2023, Vol. 10, nwac239 test) (Fig. 4B). Compared to the Y chromosome, the X chromosome exhibited a higher proportion of short-range (0.3-160 kb) cis interactions than long-range (>10 Mb) cis interactions, while the Y chromosome presented more mid-range (160 kb-10 Mb) cis interactions, indicating that the X chromosome structure is relatively denser (Fig. 4C). The conversion of Hi-C contact frequencies into Pearson correlation coefficients further showed obvious differences between the X and Y chromosomes (Fig. 4D). Compared to the segregated A and B compartments of chromosome X, the chromatin compartments of chromosome Y were divided into one large A compartment (Y1A, 0-17.85 Mb) and one large B compartment (Y1B, 19.60-40.65 Mb), with an expectedly greater number of short-range (<1.5 Mb) cis interactions in Y1B than in Y1A (P-value < 2.2 × 10 −16 , Wilcoxon test) (Supplementary Fig.  18). Moreover, we observed that the A compartment presented more trans interactions with autosomes than the B compartment on both the X (Pvalue < 2.2 × 10 −16 , Wilcoxon test) and Y (Pvalue = 6.713 × 10 −4 , Wilcoxon test) chromosomes ( Supplementary Fig. 19). Additionally, we found significant cis interactions on the Y chromosome that were obviously enriched near the SDR but were not observed on the X chromosome (Fig. 4E). To explore whether sex chromosomes maintain the stability of sex characteristics by interacting with autosomes, we identified 2389 and 3118 significant trans interactions between autosomes and the X or Y chromosome, respectively. A total of 47.5% of the significant Y-chromosome trans interactions were mapped to promoter regions, which was higher than the percentage on the X chromosome (38%). In contrast, the X chromosome showed a higher proportion (21%) of significant trans interactions that mapped to exon regions than the Y chromosome (15.6%) (Fig. 4F). Finally, the significant trans interactions of the X chromosome and Y chromosome were annotated to 1343 and 1554 genes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the genes that presented significant interactions with the X chromosome were functionally enriched in reproduction, the response to estrogen, fertilization, FoxO signaling, mTOR signaling, Notch signaling and oocyte meiosis. Furthermore, the genes that presented significant interactions with the Y chromosome were involved in male-sex differentiation, reproduction, hormone binding, gamete generation, germ-cell migration and the Wnt signaling and TGF-beta signaling pathways (Fig. 4G).

Three-dimensional chromatin reorganization coordinating with gene-expression regulation in response to sex reversal
Parallel Hi-C experiments were performed in the gonad cells of sex-reversed XX-neomale yellow catfish at the same time and using the same protocol as in XX-female and YY-supermale yellow catfish. As a result, ∼550 million read pairs and ∼254 million valid read pairs were obtained after filtering. Interestingly, we found that the Hi-C contact map of the neomale X chromosome (X M chromosome) exhibited very high similarity to the Y chromosome, with a Pearson correlation of 0.99 ( Fig. 5A and Supplementary Fig. 20). Forty-three TADs were identified on the X M chromosome, 38 (88.4%) of which shared boundaries with the Y chromosome (Fig. 5A). Similar chromosomal compartmentalization was observed between the Y chromosome and the X M chromosome; i.e. the X M chromosome is also divided into one large A compartment and one large B compartment (Fig. 5B). We then examined how contact probability depended on genomic distance on the X, X M and Y chromosomes. Compared with the X chromosome, both the X M and Y chromosomes exhibited an increased frequency of mid-range (300 kb-10 Mb) interactions and decreased frequencies of short-(<300 kb) and longrange (>10 Mb) interactions, which also indicated that the X chromosome structure was denser than those of the X M and Y chromosomes (Fig. 5C).
To verify whether similar chromatin structures correspond to similar gene expression, we compared the gonad transcriptome data of the X, X M and Y chromosomes. Both the relative gene expressions on the X M and Y chromosomes were higher than that of the X chromosome (P-value = 5.738 × 10 −11 and 4.813 × 10 −13 , Wilcoxon test), consistently with their looser chromatin structures (Supplementary Fig. 21). Only 31 differentially expressed genes (DEGs) were identified between the X M and Y chromosomes, while 498 DEGs and 540 DEGs were identified in the Y chromosome vs X chromosome and X M chromosome vs X chromosome comparisons, respectively (Supplementary Table 5). In addition, the great majority of the sex-biased DEGs identified between the Y chromosome and the X chromosome overlapped with the sex-biased DEGs between the X M chromosome and Y chromosome (male bias, 269 out of 331; female bias, 196 out of 242). This was consistent with the heat-map and hierarchical-clustering analyses performed on sexbiased genes of the X, X M and Y chromosomes, which showed a group of phenotypic males (XX males, YY supermales) clearly separated from the phenotypic female-sex-chromosome (XX females) group ( Supplementary Fig. 22).
We then investigated the local chromatin organization of the X, X M and Y chromosomes. The SDR is located in a large shared TAD (24.80-26.28 Mb) on the X M and Y chromosomes and across three small TADs on the X chromosome (Fig. 5D). Furthermore, by inspecting significant cis interactions, we observed more long-range interactions on the X M and Y chromosomes than on the X chromosome, where the SDR-related TAD contains many significant cis interactions and is located in a large loop-like domain (23)(24)(25)(26)(27)(28)(29) (Fig. 5D). Furthermore, the reconstructed 3D structures from different view angles clearly revealed the spatial differences between the X, X M and Y chromosomes (Fig. 5E and Supplementary Fig. 23). The 3D structure of the X M chromosome is more similar to that of the Y chromosome (RMSE = 1.02 × 10 −4 , spearman correlation coefficient = 0.864) compared to the X chromosome (RMSE = 1.89 × 10 −4 , spearman correlation coefficient = 0.574). The X chromosome displayed a more compact structure relative to the looser structures of the X M and Y chromosomes. In addition, the SDR could be identified in a large loop structure shared by X M and Y chromosomes, while it was not found on the X chromosome ( Fig. 5E and Supplementary Fig. 24).
To test whether sex reversal shifted the spatial arrangement of chromosomes in the nucleus, we used contact scores to assess the spatial proximity between chromosomes in XX females, XX males and YY supermales. We noted that the smaller-sized chromosomes presented more frequent trans contacts with each other, suggesting that they may be spatially close to each other ( Supplementary  Fig. 25 Fig. 26). Then, we examined the spatial proximity between the three kinds of sex chromosomes and autosomes. The spatial arrangement of the X M chromosome was highly consistent with that of the Y chromosome, with a Pearson correlation coefficient of 0.978 (P-value < 2.2 × 10 −16 ). Interestingly, the X M chromosome showed a more similar spatial arrangement to the X chromosome (Pearson correlation coefficient = 0.859, P-value = 3.788 × 10 −8 ) than to the Y chromosome (Pearson correlation coefficient = 0.778, P-value = 4.681 × 10 −6 ) ( Fig. 5F and Supplementary Fig. 27). Furthermore, the patterns of spatial proximity between the three kinds of sex chromosomes and Chromosome 8 clearly revealed stronger interactions of the X M and Y chromosomes with the autosome (Fig. 5G).

DISCUSSION
In this study, we successfully assembled highly homomorphic X and Y chromosomes by sequencing the genomes of YY-supermale and XX-female yellow catfish and discovered a new class of XX/XY sex chromosomes that originated by autosomeautosome fusion. Although the Y and X chromosomes had high sequence identity and showed no chromosome inversions or translocations, specific accumulation and distribution patterns of repetitive DNA sequences were observed on the Y chromosome. We further inferred that sex reversal could cause chromatin remodeling of X chromosomes of XX neomales and make it similar to that of the Y chromosome in YY supermales. These data might help reveal the origin and evolution of sex chromosomes and sexual plasticity in fish and amphibians. X and Y chromosomes are highly differentiated in mammals but apparently homomorphic in most fish species, such as yellow catfish, in which the sequence divergence is only <1% and gene compositions are the same between X and Y chromosomes. Rearrangements were considered as an effective way to reduce the recombination rate [49][50][51]. However, there are no classical structure features to suppress the recombination between X and Y chromosomes in yellow catfish, only minor accumulation of indels and some repetitive elements. Research on sex chromosomes without rearrangements suggested that the progressive development of recombination arrest is an alternative mechanism [52,53]. Repetitive elements, especially transposons, have been shown to play a central role in this process by causing insertion and duplication [50,[54][55][56]. A small SDR (196 kb) was identified in turquoise killifish (Nothobranchius fuzeri), in which neither inversions nor candidate sexually antagonistic genes were found, but a 241-bp deletion may be the main cause of recombination suppression [57]. Several recent studies have shown that the appearance of inversions and translocations is facilitated by the reduced recombination rates, which means the accumulation of rearrangements may be a consequence instead of a cause [11,48]. Therefore, the indels and transposons enriched in the SDR might be the main driver of initial restricted recombination suppression between young X and Y chromosomes in fish species, such as yellow catfish. Almost all sex-specific SNPs were located in the SDR of yellow catfish (Fig. 1) and 76.5% of these male-specific SNPs were located in pfpdz1, which is essential for male-sex differentiation and maintenance [58]. There are some Y-specific DNA transposons and retrotransposons in pfpdz1 gene ( Supplementary Fig. 12), while transposon-induced epigenetic modification has been shown to regulate sex determination in the fighting fish [59].
Chromosome fusion could advance the speciation process by establishing barriers to gene flow [60][61][62] and a lower chromosome number of yellow catfish compared to redtail catfish, channel catfish and southern catfish does provide evidence for chromosome fusions. Fusion between autosomes and existing Y chromosomes could create an X1 × 2Y system, with the unfused homologous segregating as a neo-X chromosome [40]. However, to date, there are no reports of females and males with different chromosome numbers in Siluriformes. Sex-chromosome turnovers in fishes are prevalent, which are usually caused by the creation of a new sex-determining gene on an autosome [63,64] or transposition of a sex-determining locus to an autosome [65,66]. Different sex chromosomes in southern catfish, channel catfish, redtail catfish and yellow catfish suggest that sex-chromosome turnovers might occurred frequently in Siluriformes (Fig. 3A). In willows, repeated turnovers lead to restricted degeneration, keeping the sex  chromosomes in a permanent state of youth [67], which might be a plausible explanation for the prevalent homomorphic sex chromosomes in Siluriformes. A HRR was observed downstream of the fusion site in yellow catfish and the SDR is located in the middle of this region with already suppressed recombination (Fig. 3). Relative to other chromosomes, there are significantly more rearrangement events in the HRR region, which were considered as a source of variation in gene diversity [68]. It has been found that the chromosomal rearrangements of the genome and the emergence of sex chromosomes are coupled in cichlids [21], which suggests that the emergence of chromosomal rearrangements could be a potential driver of neo-sex-chromosome formation. Evolutionary strata have been observed on the sex chromosomes of humans [3], chickens [69], snakes [70] and some fish including threespined stickleback [39] and spotted knifejaw [40]. In contrast, the sex chromosomes of yellow catfish did not show identifiable evolutionary strata, which indicates that the sex chromosomes of yellow catfish still remain at an early stage of sex-chromosome evolution. Subsequently, on the basis of the canonical model (Fig. 6A), we proposed two hypotheses for the evolution of sex chromosomes via chromosome fusion in yellow catfish (Fig. 6B). In the initial stages of both hypotheses, the emergence of HRRs on a pair of autosomes that resulted from several large genome-arrangement events facilitates speciation. Next, in the first hypothesis, this autosome pair might be recruited as proto-sex chromosomes after the acquisition of a sex-determining locus through diversification of a pre-existing locus in the HRR. Immediately afterwards, the transitional sex chromosomes might recruit another autosome pair to result in neo-sex chromosomes via chromosome fusion. Alternatively, the second hypothesis suggests that the emergence of proto-sex chromosomes might occur after autosome-autosome fusion. The driving forces behind these fusions are worthy of further studies. The inconsistent aspect between our hypotheses with the classical model is that the SDR-restricted recombination suppression is initiated by accumulation of repetitive elements and sex-antagonistic mutations, rather than by inversions or translocations.
Recently, epigenetic modifications, including genomic DNA methylation and histone modification, have been confirmed to control sex determination by regulating the expression of sex-determining genes in fish species and reptiles [24][25][26]. Epigenetic modifications control gene transcriptional regulation by orchestrating the 3D chromatin organization, which physically connects distant cis-regulatory elements with gene promoters through chromatin looping and compartmentalization [28,71]. However, the 3D chromatin organization of the X and Y chromosomes is unknown due to the lack of high-quality reference sequences. Although the X and Y chromosomes of yellow catfish show high sequence identity, the Y chromosome of YY supermales exhibits higher levels of chromosome organization than the X chromosome of XX females, including a larger size of TADs and a highly organized A/B compartment. The compartmental changes correspond to gene-expression levels on the X and Y chromosomes (Figs 4 and 5), which may be due to the divergent distribution of repetitive elements on the X and Y chromosomes, since repeat elements have been shown to organize the 3D genome structure and regulate gene transcription [72,73]. Chromatin loops can bring the regulatory elements and their distant targets together to a closer physical proximity [74,75]. A loop-like structure containing the SDR was observed in Y chromosome but not in X chromosome ( Fig. 5C and Supplementary Fig. 24), which might regulate the male-biased expression of candidate sex-determining genes in yellow catfish.
Sex determination in fish species and amphibians is plastic and modulated by both genetic factors (GSD) and some environmental factors (ESD), such as temperature, exogenous hormones and chemicals [22]. Moreover, environmental factors could induce epigenetic modifications and chromatin remodeling, thereby regulating patterns of gene expression [27,28,[76][77][78]. Sex reversal triggers the rapid transition from GSD to TSD in Australian bearded dragon (Pogona vitticeps) and yellow catfish [29,30]. The interaction between GSD and ESD is an important driver of sex-chromosome evolution [79]. XX-neomale yellow catfish were artificially produced by treatment with letrozole, a chemical of aromatase inhibitor [30]. The 3D chromatin organization of the sex chromosomes as well as the nucleus spatial organization of XX neomales were remodeled after sex reversal and similar to those in YY supermales. Meanwhile, gene expression and a male-specific loop containing the SDR in XX neomales are extremely similar to YY supermales. Open chromatin conformation plays a key role in the precise regulation of gene expression [28,71]. Compared with the highly condensed sex chromosomes of XX females, the open chromatin structure of X M and Y chromosomes allows the SDR and sexdetermining genes to be more easily accessed and activated (Figs 5E and 6C). Spatial organizations in the nucleus play an important role in the regulation of nuclear function [80][81][82]. Our results showed that sex chromosomes may regulate the expression of sex-related genes in different chromosomes through spatially closing to them, especially their promoters (Figs 4F and G, and 5F).
Overall, in the current study, we assembled the X and Y chromosomes of yellow catfish and provided genomic evidence that the sex-chromosome pair of the fish species is still at the initial stage of evolution. The sex chromosomes of yellow catfish originated from the fusion of two autosomes. The slight accumulation of indels and some types of repetitive elements might play an important role in the initial establishment of recombination suppression and sex-chromosome differentiation. The 3D chromatin conformation of the Y chromosome is associated with sex-specific gene expression and sex determination, while changes in the 3D chromatin structure mediated by genetic and environmental factors are suggested to drive sexual plasticity in lower vertebrates.

Fish sources
All yellow catfish used in this study were reared at the breeding centre of Huazhong Agricultural University in Wuhan City, Hubei Province. All experiments involving yellow catfish were approved and performed in compliance with the requirements of the IACUC of Huazhong Agricultural University (HZAUFI-2017-003).

Metaphase preparation and FISH
Chromosome preparation and FISH analysis were performed as previously described [83,84]. BAC clones that contain sex-linked markers have been identified and used for DNA isolation [36]. BAC DNA labeled with DIG-Nick Translation Mix (Roche) was used as the FISH probe. Cells and metaphase chromosomes on slides were denatured in 70% deionized formamide/2 × salinesodium citrate buffer (SSC) for 4 min at 72 • C. For hybridization, a 50-μl hybridization mixture containing 100 ng of labeled probe, 50% formamide, 20% dextran sulfate, 0.5 μg/μl sheared salmon sperm DNA (sssDNA), 0.1% sodium dodecyl sulfate and 2 × SSC was denatured at 95 • C for 5 min and then added to target slides and covered with a 24 × 50 mm 2 coverslip to spread the hybridization solution. The hybridization reaction was performed in a wet box at 37 • C for 24-48 hours. After a series of post-hybridization washes, the slides were co-incubated with a fluorescein isothiocyanate (FITC)-conjugated anti-digoxigenin antibody (Roche) and diamidino-phenyl-indole (DAPI) for 1 hour, and the fluorescent signals were detected and captured under a ×63 oil lens.

Experimental procedures and data analysis of genome sequencing, Hi-C and RNA-seq
Detailed descriptions of experimental procedures and data analysis are available in the Supplementary data.

Quantitative real-time PCR (qRT-PCR)
After determining the genotypic sex of XX female, XX neomale and YY supermale by sex-linked markers [36], an XX-female population and an XY-male population were produced by crossing XX males and YY supermales with XX females. Gonads of 30